Supplementary Information

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1 Supplementary Information Minimal mechanistic model of sirna-dependent target RNA slicing by recombinant human Argonaute2 protein Andrea Deerberg*, Sarah Willkomm* and Tobias Restle Institute of Molecular Medicine, Universitätsklinikum Schleswig-Holstein, University of Lübeck, Ratzeburger Allee 16, Lübeck, Germany. Correspondence should be addressed to T.R. *These authors contributed equally to this work. Supplementary Figure 1: SDS-PAGE analysis of purified recombinant GST-hAgo2.

2 A B Supplementary Figure 2: Graphical illustration of the different fluorescently labeled RNA substrates used in the present study. Strands are shown in 5' - 3' orientation. (A) Different guide or target strands including position of the fluorophores/quencher. (B) Guide/target combinations for analyzing ternary complex formation and product release. Sequences are given in table 1. Direction of fluorescence change is indicated by little arrows. Deerberg et al. 2

3 Supplementary table 1: Summary of all stopped flow experiments performed in the present study. Binary complexes Content syringe 1 Content syringe 2 k +1_bin (M -1 s -1 ) collision complex *alternatively k +1_bin_obs (s -1 ) k +2_bin (s -1 ) guide 5 -end binding to MID k +3_bin (s -1 ) guide 3 -end binding to PAZ Remark Exp. 1 guide binding (5 -phosphorylated guide) hago2 as2b FAM.6 x see Fig. 2A Exp. 2 guide binding (5 -unphosphorylated guide) hago2 OH-as2b FAM.6 x Exp. 3 - guide binding (fluorophore located at the 5'-position) hago2 aslam FAM 13 s -1* - - Exp. 4 - guide binding (fluorophore located at the 3'-position) hago2 slam Alexa s -1*.1.1 Exp. 5 - guide binding (19mer) hago2 19mer FAM 37 s -1*.26.1 Exp. 6 guide binding (5 -phosphorylated guide) to hago2-paz9 hago2-paz9 as2b FAM.2 x Exp. 7 sirna binding (double-strand RNA) hago2 as2b FAM /s2b 1.2 x Content Content k -1_bin (s -1 ) k -2_bin (s -1 ) k -3_bin (s -1 ) syringe 1 syringe 2 guide dissociation guide 5 -end release from MID guide 3 -end release from PAZ 2 nd and 3 rd phase not observable PAZ domain binding defective see Fig. S13A Remark Exp. 8 guide dissociation (5 -phosphorylated guide) unlabeled competitor see Fig. S5 as2b FAM RNA Exp. 9 guide dissociation (5 -unphosphorylated guide) unlabeled competitor as2b FAM RNA Exp. 1 guide dissociation (5 -phosphorylated guide) from hago2-paz9 unlabeled competitor hago2-paz and as2b FAM RNA Exp. 11 sirna dissociation (double-strand RNA) as2b FAM /s2b unlabeled competitor RNA PAZ domain binding defective see Fig. S13B Deerberg et al. 3

4 Supplementary table 1 continued Ternary complexes Content Content syringe 1 syringe 2 k +1_ter (M -1 s -1 ) collision complex *alternatively k +1_ter_obs (s -1 ) k +2_ter (s -1 ) seed pairing k +3_ter (s -1 ) guide 3 -end release from PAZ Remark Exp. 12 target binding (complementary target) as2b FAM 3.2 x see Fig. 2B s2b Exp. 13 target binding (complementary target) to binary complex with hago2-paz9 hago2-paz9 s2b 2.9 x and as2b FAM Exp. 14 target binding (seed mismatched target) as2b FAM s2b mm-5' 43 s -1* -.1 PAZ domain binding defective see Fig. S13C see Fig. S8A 2 nd phase not observable Exp. 15 target binding (3 -end mismatched target) as2b FAM s2b mm-3' 12 s -1*.3 - see Fig. S8B 3 rd phase not observable Exp target binding (mismatched target) asgl3 Content syringe 1 as2b FAM 24 s -1* - - Content syringe 2 k -1_ter (s -1 ) target dissociation k -2_ter (s -1 ) seed dissociation k -3_ter (s -1 ) 3'-guide/5'- target dissociation 2 nd and 3 rd phase not observable Remark Exp. 17 target dissociation (competition with unlabeled guide) as2b FAM /s2b as2b see Fig. S7A Exp. 18 target dissociation from ternary complex with hago2-paz9 hago2-paz9 and as2b FAM /s2b as2b PAZ domain binding defective see Fig. S13D Exp. 19 target dissociation (competition with unlabeled target) as2b see Fig. S7B s2b/as2b FAM Exp. 2 dissociation of cleaved target (competition with unlabeled guide) s2b FAM /as2b see Fig. S12 note: s2b.3 measured with fluorescence spectrometer *concentration dependency not measured Deerberg et al. 4

5 Supplementary Figure 3: hago2 with modeled 2-mer sirna duplex. The sirna (pdb: 1R9F) was roughly aligned with the intrinsically bound guide strand (pdb: 4EI1) and subsequently adjusted manually to minimize steric clashes using the program UCSF Chimera. N-terminal, PAZ, Mid and PIWI domains are color coded blue, magenta, gold and green, respectively. The two linker regions are shown in grey. Guide RNA is shown in yellow and target RNA in red. Position 14 (5'-end equivalent to position 1) of the guide strand is shown in black. The C 6 -linked 5/6-FAM at position 14 is shown in orange. Deerberg et al. 5

6 8 6 k1, obs (s -1 ) hago2 (nm) Supplementary Figure 4: Concentration dependence of the first phase of guide RNA binding (observed pseudo-first-order rate constant) with hago2 during binary complex formation is shown. A constant concentration of fluorescently labeled guide RNA (as2b FAM, 5 nm) was mixed with increasing amounts of hago2. k +1_bin and k -1_bin were determined by the slope of the linear fit and the intercept of the line with the y-axis and yielded values of.6 x 1 8 M -1 s -1 and 6.2 (±.6) s -1, respectively. Deerberg et al. 6

7 1,2 relative fluorescence 1,1 1, Supplementary Figure 5: Pre-steady-state kinetic analysis of the dissociation of binary complexes. A typical stopped-flow result is shown. Guide RNA (as2b FAM ; 2 nm) was preincubated with hago2 (3 nm) and then rapidly mixed with a 1-fold excess of non-labeled competitor guide RNA. The change in fluorescence signal was detected over time to determine dissociation rate constants. The best fit of the experimental data to a triple exponential equation yielded rates of k -1_bin : 14.7 (± 2.5) s -1, k -2_bin :.17 (±.2) s -1 and k -3_bin :.6 (± 8x1-5 ) s -1. Deerberg et al. 7

8 5 4 k1, obs (s -1 ) s2b BHQ (nm) Supplementary Figure 6: Concentration dependence of the first phase of target RNA binding (observed pseudo-first-order rate constant) with hago2 during ternary complex formation is shown. A constant concentration of fluorescently labeled guide RNA (as2b FAM ; 5 nm) bound to hago2 was mixed with increasing amounts of target (s2b BHQ ) RNA. k +1_ter and k -1_ter were determined by the slope of the linear fit and the intercept of the line with the y-axis and yielded values of 3.2 x 1 8 M -1 s -1 and 11.8 (± 2.) s -1, respectively. Deerberg et al. 8

9 A,2 relative fluorescence -,2 -, , B relative fluorescence,4 -,4 -,8,4,4, Supplementary Figure 7: Pre-steady-state kinetic analysis of the dissociation of ternary complexes using either guide or target strands as competitor. Typical stoppedflow results are shown. Stopped-flow measurement of the competition of a ternary complex [hago2 (6 nm), guide RNA (as2b FAM ; 2 nm) and target RNA (s2b; 6 nm)] or [hago2 (6 nm), guide RNA (s2b; 2 nm) and target RNA (as2b FAM ; 6 nm)] with a 33-fold excess of unlabeled guide (A) or target RNA (B). Experimental data were fitted to a triple exponential equation. The results are listed in supplementary table 2. The inserts show the reactions on a shorter time scale. Deerberg et al. 9

10 A B C Supplementary Figure 8: Pre-steady-state kinetic analysis of ternary complexes with mismatched guide and target strands. Typical stopped-flow results are shown. (A) Stopped-flow measurements of the assembly of a ternary complex with s2b mm-5' RNA resulting in a mismatched seed-region. (B) Stopped-flow measurements of the assembly of a ternary complex with s2b mm-3' RNA resulting in a mismatched 3 -region. In both cases binary hago2-guide RNA (as2b FAM ) complexes (6 and 2 nm) were rapidly mixed with the corresponding target RNA (6 nm). Experimental data were fitted best to a double exponential equation. The results are listed in supplementary table 3. The inserts show the reactions on a shorter time scale. (C) Cartoon illustrating matching and non-matching guide and target RNA regions. Guide is shown in orange and target in blue. Deerberg et al. 1

11 A B Supplementary Figure 9: hago2-mediated cleavage of target RNA. Cleavage assays were conducted using 3.7 µm hago2, 1 nm guide RNA (as2b) and 2.5 nm target RNA (ICAM-1-IVT). Reactions were analyzed by 8% (w/v) PAGE. (A) Samples were collected at time points to 3. (B) Samples were collected at, 3, 6 and 12. Deerberg et al. 11

12 25 cleavage (%) Supplementary Figure 1: hago2-catalyzed target RNA cleavage reactions in presence or absence of a trap (competitor RNA). 2.5 µm 1 nm as2b were preincubated in cleavage buffer with.5 mm Mg ++ for 5 minutes, then 2.5 nm 5-32 P-labelled s2b was added and incubated for another 5 minutes. Cleavage reaction was started by increasing the Mg ++ concentration to 2 mm in the presence (closed circles) or absence (open circles) of 1 µm competitor RNA (ICAM-1-IVT-Reverse) preventing multiple turnover by trapping dissociated target RNA. To exclude protein aggregation potentially induced by high concentrations of competitor nucleic acid, ICAM-1-IVT-Reverse was replaced by 1 µm 15- mer poly(ra)-rna (squares) in a control reaction. Samples were taken at time points given, analyzed by denaturing PAGE and visualized via autoradiography. Fitting of the experimental data of the cleavage reaction in absence of competitor RNA or in case of poly(ra)-rna to a single exponential equation yielded rate constants of.5 (±.1) and.5 (±.6) s -1, respectively. The corresponding fitted amplitudes are 9.6 (± 17.1) and 88.3 (± 8.8) %. In presence of competitor RNA no cleavage could be detected. Sequence of ICAM-1- IVT-Reverse: ggg cga auu ggg ccc ucu aga ugc aug cuc gag cgg ccg cca gug uga ugg aua ucu gca gaa uuc gcc cuu uau uuc uug auc uuc cgc ugg cgg uua uag agg uac gug cug agg ccu gca gug ccc auu aug acu gcg gcu aag ggc gaa uuc cag cac acu ggc ggc cgu uac uag ugg auc cga gcu. The s2b matching sequence in ICAM-1-IVT-Reverse is underlined. Deerberg et al. 12

13 4 cleavage (%) Supplementary Figure 11: hago2-catalyzed passenger RNA cleavage reaction in presence of ICAM-1-IVT. Cleavage reaction was started by adding 1 nm ds sirna hybrid (passenger strand 5-32 P-labelled) and 5-32 P-labelled ICAM-1-IVT to 2.5 µm cleavage buffer containing 2 mm Mg ++. Samples were taken after different time points, analyzed by denaturing PAGE and visualized via autoradiography. Fitting of the experimental data to a single exponential equation yielded rate constants of.4 (±.5) s -1 for the passenger strand cleavage (open circles) and.1 (±.2) s -1 for the ICAM-1-IVT cleavage (closed circles). The corresponding fitted amplitudes are 5% for passenger cleavage and 37% for ICAM-1-IVT cleavage. Deerberg et al. 13

14 8 relative fluorescence Supplementary Figure 12: Product release represents the overall rate limiting step. Guide RNA (s2b FAM ; 4 nm) and target RNA (as2b; 4 nm) were pre-annealed and preincubated with hago2 (3 µm) for 2h at 37 C in presence of 2 mm Mg ++ to allow formation of ternary complexes and subsequent accumulation of target RNA cleavage products. This was followed by mixing with a 1-fold excess of non-labelled competitor guide RNA. The change in fluorescence signal was detected over time and the rate of product release was determined by fitting the experimental data to an exponential equation yielding a rate of.3 (± 1.7 x 1-5 ) s -1 (equivalent to k +8 in scheme 1). Deerberg et al. 14

15 A,86 B 1,7 relative fluorescence,84,82,8,78,9,85,8,4,8 relative fluorescence 1,6 1,5 1,4 1,3 1,6 1,55,5 1,76 1,2 C D ,62 relative fluorescence -,4 -,6 -,8-1 -1,4-1,8,5 1 relative fluorescence,6,58,56,65,6,55,5 1 E binary complex k +1_bin (M -1 s -1 ) k -1_bin (s -1 ) k +2_bin (s -1 ) k -2_bin (s -1 ) K d_cal (nm) as2b FAM.2 x ternary complex k +1_ter (M -1 s -1 ) k -1_ter (s -1 ) k +2_ter (s -1 ) k -2_ter (s -1 ) K d_cal (nm) s2b 2.9 x Supplementary Figure 13: Pre-steady-state kinetic analysis of binary and ternary complex formation with hago2-paz9. Typical graphs are shown. (A) Association of binary complexes. hago2-paz9 (5 nm) was rapidly mixed with guide RNA (as2b FAM ; 2 nm). (B) Dissociation of binary complexes. hago2-paz9 was pre-incubated with guide RNA (as2b FAM ; 2 nm) and then rapidly mixed with a 1-fold excess of unlabeled guide RNA (as2b; 2 µm). (C) Association of ternary complexes. hago2-paz9 was pre-incubated with guide RNA (as2b FAM ; 2 nm) and then rapidly mixed with target RNA (s2b; 4 nm). (D) Dissociation of ternary complexes. hago2-paz9 was pre-incubated with guide RNA (as2b FAM ; 2 nm) and target RNA (s2b; 4 nm). Preformed ternary complexes were then rapidly mixed with a 1-fold excess of unlabeled guide RNA (as2b; 2 µm). In all four cases the change of fluorescence was recorded over time. Rate constants were determined by fitting the data to a double exponential equation. The results of these fits are shown in (E). Deerberg et al. 15

16 Supplementary Table 2: Target dissociation rate constants using either guide or target RNA as competitor. See supplementary figure 7 for details. Competition with guide RNA Competition with target RNA k -1_ter 1.2 (±.3) s (± 6) s -1 k -2_ter.21 (±.1) s -1.2 (±.4) s -1 k -3_ter.2 (± 5.8 x 1-5 ) s (±.4) s -1 Supplementary Table 3: Association rate constants for complementary and partially mismatched target RNAs. See supplementary figure 8 for details. sirna k +1_obs_ter k +2_ter k +3_ter complementary 33 (± 7.5) s -1.1 (±.3) s -1.3 (± 2x1-5 ) s -1 5'-mismatch 43 (± 15) s (±,3) s mismatch 12 (± 1.) s -1.3 (±.2) s -1 - Deerberg et al. 16